Project Summary Dyneins are AAA+ motors responsible for minus-end-directed motility along microtubules (MTs) and play fundamental roles in cargo transport, mitosis, and ciliary beating. Dynein is currently the focus of the motor field as the mechanism of its movement is not well understood in comparison to plus-end-directed kinesins. Despite rapid transport of dynein-driven cargos in cells, previous in vitro studies identified mammalian dynein as a weak motor, exhibiting slow motility and producing lower forces than kinesin. Recently, in vitro reconstitution of the dynein-dynactin machinery revealed that mammalian dynein is autoinhibited when not transporting cargo, and motility is activated when dynein forms a 2.5 MDa ternary complex with its cofactor dynactin and a cargo binding adaptor. Therefore, all of the previous in vitro work on mammalian dynein used inactive motor and their conclusions do not reflect how active dynein-dynactin machinery transports cargos in cells. Our future goals are to dissect the mechanism of active cytoplasmic dynein complexes and determine how dynein activation and motility are regulated across multiple scales using single molecule imaging, optical trapping, MD simulations, and cryoEM. Specifically, we will determine how Lis1 plays a role in the activation of and regulation of dynein motility. We will also study dynein motility in physiologically relevant conditions and ask whether MT-associated proteins, MAP7 and Tau, inhibit dynein motility by sterically blocking its tubulin binding site or by excluding its MT binding via liquid-liquid demixing. We will also characterize the motility of dynein and dynactin disease mutants to reveal the molecular mechanism of neuropathies associated with these mutations. Finally, we will reconstitute the entire MT transport machinery using cargo adaptors identified by in vivo studies of mitochondria, autophagosomes, and vesicle transport, but not yet characterized in vitro. Using this approach, we will dissect how cargo adaptors regulate motors to control the bidirectional transport of these cargos. We will also study ciliary dyneins that slide parallel array of axonemal MTs to power ciliary beating. Several models have been proposed to explain how the sliding activity of dyneins is self-regulated to orchestrate ciliary oscillations. Predictions that these models make about the mechanism of ciliary dyneins have not been directly tested. Recently, a recombinant expression system was developed for Tetrahymena outer-arm dynein (OAD), enabling us to perform in-depth structural and biophysical studies of ciliary dyneins. Unlike cytoplasmic dynein, OAD forms a heterodimer and is not processive. Using this system, we will characterize the mechanism of OAD motility and force generation. We will then directly test the predictions of each model by constructing in vitro geometries that mimic dynein/MT interactions in a beating cilium. Finally, we will identify structural components that give rise to the nonprocessive motility, curvature sensing, and self-oscillatory behavior of OAD. The success of our research program will reveal the fundamental mechanochemistry of dynein and how it achieves retrograde transport of intracellular cargos and drives the self-coordinated oscillations of motile cilia.